We have previously shown that supernatants derived from natural killer (NK) cells of HIV-seronegative individuals suppress HIV replication in human monocytes/macrophages (MO) and monocytic cell lines, but not in human T lymphocytes or T cell lines. Experiments have been undertaken to determine the nature of the factor(s) responsible for this suppression. Our results, thus far, show that the NK cell-derived supernatant contains interferon-gamma (IFN-gamma) and granulocyte/monocyte colony stimulating factor (GM-CSF), and that each of these cytokines alone can suppress viral replication. Sequential removal of IFN-gamma (17 kD) and GM-CSF (23 kD) from the supernatant using immunoaffinity gels did not eliminate the suppressive activity. Size exclusion analysis revealed that the NK cell-derived suppressor factor has an apparent molecular weight greater than 30 kD. An equally suppressive supernatant can be generated using the human NK cell line, NK3.3 When this latter supernatant is subjected to monosulfate column chromatography, HIV suppressive activity is present in both the flow through and the eluted fractions. However, GM-CSF and IFN-gamma are only detectable in the flow through, not the eluated, fraction. These data indicate that NK cells synthesize a greater than 30 kD HIV-suppressive factor distinct from IFN-gamma and GM-CSF. Work is in progress to identify and characterize this potentially novel factor.